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Roux-en-Y gastric bypass (RYGB), the most effective surgical procedure for weight loss, decreases obesity and ameliorates comorbidities, such as non-alcoholic fatty liver (NAFLD) and cardiovascular (CVD) diseases. Cholesterol is a major CVD risk factor and modulator of NAFLD development, and the liver tightly controls its metabolism. How RYGB surgery modulates systemic and hepatic cholesterol metabolism is still unclear. We studied the hepatic transcriptome of 26 patients with obesity but not diabetes before and 1 year after undergoing RYGB. In parallel, we measured quantitative changes in plasma cholesterol metabolites and bile acids (BAs). RYGB surgery improved systemic cholesterol metabolism and increased plasma total and primary BA levels. Transcriptomic analysis revealed specific alterations in the liver after RYGB, with the downregulation of a module of genes implicated in inflammation and the upregulation of three modules, one associated with BA metabolism. A dedicated analysis of hepatic genes related to cholesterol homeostasis pointed towards increased biliary cholesterol elimination after RYGB, associated with enhancement of the alternate, but not the classical, BA synthesis pathway. In parallel, alterations in the expression of genes involved in cholesterol uptake and intracellular trafficking indicate improved hepatic free cholesterol handling. Finally, RYGB decreased plasma markers of cholesterol synthesis, which correlated with an improvement in liver disease status after surgery. Our results identify specific regulatory effects of RYGB on inflammation and cholesterol metabolism. RYGB alters the hepatic transcriptome signature, likely improving liver cholesterol homeostasis. These gene regulatory effects are reflected by systemic post-surgery changes of cholesterol-related metabolites, corroborating the beneficial effects of RYGB on both hepatic and systemic cholesterol homeostasis. Roux-en-Y gastric bypass (RYGB) is a widely used bariatric surgery procedure with proven efficacy in body weight management, combatting cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD). RYGB exerts many beneficial metabolic effects, by lowering plasma cholesterol and improving atherogenic dyslipidemia. Using a cohort of patients undergoing RYGB, studied before and 1 year after surgery, we analyzed how RYGB modulates hepatic and systemic cholesterol and bile acid metabolism. The results of our study provide important insights on the regulation of cholesterol homeostasis after RYGB and open avenues that could guide future monitoring and treatment strategies targeting CVD and NAFLD in obesity. [Display omitted] • Body weight management by RYGB surgery has been shown to ameliorate both CVD and NAFLD. • We assessed the hepatic gene expression and plasma metabolite changes in patients after RYGB surgery. • Expression of genes of the alternate BA synthesis and biliary cholesterol elimination pathways are induced after RYGB. • RYGB increases plasma FGF19 and lowers plasma C4 indicating decreased BA synthesis through the classical pathway. • Decreased plasma cholesterol synthesis markers upon RYGB correlate with improved NAFLD histology after surgery. [ABSTRACT FROM AUTHOR]

Over the last decade, immunometabolism has emerged as a novel interdisciplinary field of research and yielded significant fundamental insights into the regulation of immune responses. Multiple classical approaches to interrogate immunometabolism, including bulk metabolic profiling and analysis of metabolic regulator expression, paved the way to appreciating the physiological complexity of immunometabolic regulation in vivo. Studying immunometabolism at the systems level raised the need to transition towards the next-generation technology for metabolic profiling and analysis. Spatially resolved metabolic imaging and computational algorithms for multi-modal data integration are new approaches to connecting metabolism and immunity. In this review, we discuss recent studies that highlight the complex physiological interplay between immune responses and metabolism and give an overview of technological developments that bear the promise of capturing this complexity most directly and comprehensively. [ABSTRACT FROM AUTHOR]

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ‐dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ‐dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide–treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract: Complement C3 is involved in various protective and regulatory mechanisms of immune system. Recently it was established that C3 expression is regulated by nuclear receptors. Hepatic nuclear factor 4α (HNF4α) is a nuclear receptor critical for hepatic development and metabolism. We have shown that HNF4α is a positive regulator of C3 gene expression, realizing its effects through binding to two HNF4-response elements within the C3 promoter in HepG2 cells. TNFα is a well established positive regulator of C3 expression in hepatocytes during acute phase of inflammation. TNFα decreases the amount of HNF4α protein in HepG2 cells through NF-κB and MEK1/2 pathways thereby leading to a decrease in HNF4α bound to the C3 promoter. TNFα and HNF4α act in a synergetic way resulting in the potent activation of C3 transcription. These results suggest a novel mechanism of C3 regulation during acute phase response in HepG2 cells and display the mechanism of interaction of TNFα-induced pathways and HNF4α in transcriptional regulation of C3 gene. [Copyright &y& Elsevier]

Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor α (PPARα) in human hepatoma HepG2 cells and mouse liver. Using PPARα siRNA and synthetic PPARα agonist WY-14643 and antagonist MK886 we showed that activation of PPARα results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPARα in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse and WY-14643 stimulates mouse C3 expression in the liver. Tiffα increases C3 gene via NF-κB and, to a lesser extent, MEK1/2 signaling pathways, whereas TNFα-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK In HepG2 cells. Activation of PPARα abolishes TNFα-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-κB via PPRE-dependent mechanism in HepG2 cells. TNFα decreases PPARα protein content via NF-κB and MEK1/2 signaling pathways and inhibits PPARα binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPARα and TNFα in the regulation of complement system. [ABSTRACT FROM AUTHOR]

Apolipoprotein A-I (ApoA-I) is the main functional protein component of human high-density lipoproteins. ApoA-I shows various anti-inflammatory and atheroprotective properties toward macrophages; however, endogenous apoA-I expression has not been investigated in macrophages. We have shown that endogenous apoA-I gene is expressed in human macrophages at both mRNA and protein levels. Endogenous ApoA-I is localized in intracellular vesicles and at the external side of the plasma membrane in association with ATP-binding cassette transporter A1 (ABCA1) and lipid rafts in macrophages. We have shown that endogenous ApoA-I stabilizes ABCA1, moreover, down-regulation of ApoA-I by siRNA results in an increase of Toll-like receptor 4 (TLR4) mRNA and membrane surface protein expression, as well as an enhancement of bacterial lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and inducible nitric oxide synthase (NOS2) genes in human macrophages. TNF-α stimulates ApoA-I expression and secretion (1.2±0.2 vs. 4.3±0.9 ng/mg total protein) in macrophages. Obtained results suggest that endogenous ApoA-I has anti-inflammatory properties, presumably due to ABCA1 stabilization in macrophages; these results elucidate the cell type-specific mechanism of the TNF-α-mediated regulation of apoA-I gene expression in monocytes and macrophages. [ABSTRACT FROM AUTHOR]

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liverXreceptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRβ in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3 a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mL DL, C3, C3a, and TLR4 during development of atherosclerotic lesions. [ABSTRACT FROM AUTHOR]

Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPARγ is known as activator of ABCA1 expression, but details of PPARγ-mediated regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPARγ activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXRβ binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1/LXRβ complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex, but does not block PPARγ-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPARγ may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPARγ, LXRβ and MEK1/2 in regulation of ABCA1 mRNA and protein expression. [ABSTRACT FROM AUTHOR]

Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5′-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching. [Copyright &y& Elsevier]

The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. In this work, we have demonstrated that treatment of HepG2 human hepatoma cells with chemical inhibitors for JNK, p38 protein kinases, and NFκB transcription factor abolishes the TNFα-mediated inhibition of human apoA-I gene expression in HepG2 cells. In addition, we have shown that TNFα decreases also the rate of secretion of apoA-I protein by HepG2 cells, and this effect depends on JNK and p38, but not on NFκB and MEK1/2 signaling pathways. The inhibitory effect of TNFα has been found to be mediated by the hepatic enhancer of the apoA-I gene. The decrease in the level of human apoA-I gene expression under the impact of TNFα appears to be partly mediated by the inhibition of HNF4α and PPARα gene expression. Treatment of HepG2 cells with PPARα antagonist (MK886) or LXR agonist (TO901317) abolishes the TNFα-mediated decrease in the level of apoA-I gene expression. PPARα agonist (WY-14643) abolishes the negative effect of TNFα on apoA-I gene expression in the case of simultaneous inhibition of MEK1/2, although neither inhibition of MEK1/2 nor addition of WY-14643 leads to the blocking of the TNFα-mediated decrease in the level of apoA-I gene expression individually. The ligand-dependent regulation of apoA-I gene expression by PPARα appears to be affected by the TNFα-mediated activation of MEK1/2 kinases, probably through PPARα phosphorylation. Treatment of HepG2 cells with PPARα and LXR synthetic agonists also blocks the inhibition of apoA-I protein secretion in HepG2 cells under the impact of TNFα. A chromatin immunoprecipitation assay demonstrates that TNFα leads to a 2-fold decrease in the level of PPARα binding with the apoA-I gene hepatic enhancer. At the same time, the level of LXRβ binding with the apoA-I gene hepatic enhancer is increased 3-fold under the impact of TNFα. These results suggest that nuclear receptors HNF4α, PPARα, and LXRs are involved in the TNFα-mediated downregulation of human apoA-I gene expression and apoA-I protein secretion in HepG2 cells. [ABSTRACT FROM AUTHOR]

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8+ T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK+CD8+ T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK+ Taa cells as a potential target to address age-associated dysfunctions of the immune system. • scRNA/CITE-seq and scATAC-seq reveal hallmarks of immune aging in mice and humans • Clonal exhausted-like GZMK+CD8+ T cells accumulate in tissues due to old environment • GZMK secreted by activated GZMK+CD8+ T cells can promote SASP from senescent cells • GZMK+CD8+ Taa rather than GZMB+CD8+ Tem accumulate in human blood with age Aging affects the immune system and establishes a chronic low-grade inflammation (inflammaging). Mogilenko et al. defined organ-specific and common alterations of immune cell populations in aging and identified a distinct subset of clonal GZMK+ CD8+ T cells as a conserved cellular hallmark of inflammaging in mice and humans. [ABSTRACT FROM AUTHOR]

Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR. • Extracellular FAs inhibit HK activity and rewire metabolism in late TLR activation • FA-mediated adaptation of glycolysis leads to perturbed mitochondrial fitness • Metabolic adaptation and mitochondrial ROS exacerbate TLR-induced UPR activation • The UPR links metabolic alterations to a distinct inflammatory signature A high-fat diet induces the metabolic rewiring of TLR-activated dendritic cells and exacerbates IL-23-mediated psoriatic skin inflammation. [ABSTRACT FROM AUTHOR]

Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and β prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression. [ABSTRACT FROM AUTHOR]

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25–85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK +CD8+ T cells and HLA-DR +CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C + GZMB − CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4 +CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource. [Display omitted] • Clinical and scRNA-seq data of 2 million PBMCs from 166 people aged 25–85 years old • Type 2 memory CD4+ and CD8+ T cells increase with age and can produce more IL-4 • NKG2C+GZMB–XCL1+ defines a unique memory CD8+ T cell subset that decreases with age • HLA-DR+ CD4+ memory T cells and GZMK+ CD8+ T cells accumulate with age Terekhova et al. perform single-cell RNA sequencing on >300 samples between 25 and 85 years to comprehensively profile immune changes throughout aging. This resource revealed 12 subpopulations that change with age, including increased type 2 memory CD4+ and CD8+ T cells and decreased NKG2C+GZMB−CD8+ memory population. [ABSTRACT FROM AUTHOR]

The article presents the discussion on investigating the origin and dynamics of meningeal myeloid cells, a three-layer membrane covering the central nervous system (CNS) corroborated the finding by flow cytometry and confocal imaging.